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Originally published In Press as doi:10.1074/jbc.M112010200 on January 18, 2002

J. Biol. Chem., Vol. 277, Issue 15, 12587-12595, April 12, 2002
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De Novo Ceramide Regulates the Alternative Splicing of Caspase 9 and Bcl-x in A549 Lung Adenocarcinoma Cells

DEPENDENCE ON PROTEIN PHOSPHATASE-1*

Charles E. ChalfantDagger §, Kristin RathmanDagger , Ryan L. PinkermanDagger , Rachel E. WoodDagger , Lina M. ObeidDagger §, Besim OgretmenDagger , and Yusuf A. HannunDagger ||

From the Dagger  Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, § Research and Development, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29401, and the  Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425

Received for publication, December 17, 2002, and in revised form, January 15, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have demonstrated that several splice variants are derived from both the caspase 9 and Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C6 ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose- and time-dependent manner. Pretreatment with calyculin A (5 nM), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nM), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 µM) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nM), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine down-regulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C6 ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo pathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulators of apoptosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apoptosis (used here interchangeably with programmed cell death) is a mechanism (or group of mechanisms) by which cells execute endogenous programs of cell death, often in response to adverse external or internal signals or sources of injury (1-4). In the case of cancer, interference with this system of programmed cell death can lead to expansion of deleterious cells. Apoptosis is regulated by multiple factors through complex mechanisms (3, 4). It has become well established that many inducers of apoptosis activate caspases, and that the activation of these proteases is perhaps the point of irreversible commitment to the onset of apoptosis (5). Furthermore, the Bcl-2 family has also been widely implicated in regulating apoptotic machinery (6-9).

Several factors that regulate apoptosis have splice variants with an opposite/dominant negative function. The anti-apoptotic factor, Bcl-x(L), was the first shown to have a dominant negative splice variant, Bcl-x(s) (10). Dominant-negative splice variants of two members of the caspase family, caspase 2(s) and caspase 9b, have now also been described with both inhibiting apoptosis induced by most chemotherapeutic agents and extracellular agonists (11-13). Several splice variants of Bax have been described with the most recent, Baxomega , shown to block tumor necrosis factor alpha -induced apoptosis (14-19). Although recognized for metabolic and mitogenic pathways, the importance of alternative splicing in apoptosis and its mechanisms of regulation have been overlooked and largely unstudied.

In other lines of investigation, studies have led to the identification of ceramide as a potential inducer and mediator/regulator of apoptosis in response to tumor necrosis factor alpha  and many chemotherapeutic agents (20-31). More recent studies have begun to relate the action of ceramide to Bcl-2 and death caspases (20-25, 32, 33).

While searching for direct targets of ceramide, a ceramide-activated protein phosphatase (CAPP) was identified. To date, two families of protein phosphatases, protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A), have been shown to be activated by ceramide in vitro (34-37). With the demonstration of PP1 as a ceramide-activated protein phosphatase, potential PP1 substrates and mechanisms regulated by PP1 became candidate targets for ceramide action.

SR proteins, a family of arginine/serine-rich domain containing proteins and specific PP1 substrates, are required for constitutive and alternative pre-mRNA processing (38-48). Endogenous ceramide has recently been found to modulate the phosphorylation status of SR proteins in a PP1-dependent manner (49). Several reports have also demonstrated a role for PP1 in regulating alternative splicing, and two spliceosomal targeting subunits for PP1 have been described (38, 48, 50, 51). Therefore, PP1 may play a role in regulating RNA processing in response to stimuli, in particular, it may define a pathway linking ceramide to the regulation of the alternative splicing of apoptosis regulators.

In this study, endogenous ceramides produced via the de novo sphingolipid pathway are shown to regulate the alternative pre-mRNA processing of caspase 9 and Bcl-x pre-mRNA. Furthermore, this novel and newly defined mechanism is shown to be mediated by protein phosphatase-1.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- A549 adenocarcinoma cells were grown in a 50% RPMI 1640 (Invitrogen) and 50% Dulbecco's modified Eagle's medium (Invitrogen) mixture supplemented with L-glutamine, 10% (v/v) fetal bovine serum (Sigma), 200 units/ml penicillin G sodium, and 200 µg/ml streptomycin sulfate. Cells were maintained at less than 80% confluency under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C). For treatments with D-e-C6 ceramide, A549 cells were plated at 4 × 105 cells/35-mm plate in RPMI 1640 and Dulbecco's modified Eagle's medium supplemented with 2% (v/v) fetal bovine serum.

RNA Extraction-- Total RNA was extracted using Trizol reagent (Invitrogen). The sample was then separated into an upper aqueous phase and a lower organic phase by centrifugation at 12,000 × g for 15 min at 4 °C. The aqueous phase was transferred to a new tube and the RNA was precipitated by the addition of 1 volume of isopropyl alcohol. The precipitant was incubated for 10 min at room temperature, and RNA was collected by centrifugation at 12,000 × g for 15 min at 4 °C. The pellet was washed with 1 ml of 75% ice-cold ethanol, and the RNA was resuspended in nuclease-free water for storage at -80 °C.

Reverse Transcriptase-Polymerase Chain Reaction (RT1-PCR)-- For both Bcl-x and caspase 9 analysis, 1 µg of total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo(dT) as the priming agent. After 1 h incubation at 43.5 °C, the reactions were stopped by 70 °C heating for 15 min. Template RNA was then removed using RNase H (Invitrogen).

For evaluating Bcl-x splice variant expression, an upstream 5' primer to Bcl-x (5'-GAGGCAGGCGACGAGTTTGAA-3') and a 3' primer (3'-TGGGAGGGTAGAGTGGATGGT-5') were used. Using these primers, 10% of the reverse transcriptase reaction was amplified for 35 cycles (94 °C, 30s; 58 °C, 30 s; 72 °C, 1 min) using Platinum Taq DNA polymerase (Invitrogen).

For evaluating caspase 9 splice variant expression, an upstream 5' primer to caspase 9 (5'-GCTCTTCCTTTGTTCATCTCC-3') and a 3' primer (5'-CATCTGGCTCGGGGTTACTGC-3') were used. Using these primers, 10% of the reverse transcriptase reaction was amplified for 35 cycles (94 °C, 30s; 58 °C, 30 s; 72 °C, 1 min) using Platinum Taq DNA polymerase (Invitrogen).

Construction and Labeling of Riboprobes-- The caspase 9 and caspase 9b riboprobe templates were constructed by cloning the RT-PCR fragments for caspase 9 (724 bp) and caspase 9b (274 bp) into pCR-Blunt II-TOPO vector. Constructs were verified for orientation and the lack of mutagenesis by DNA sequencing. To produce caspase 9 cRNA, the riboprobe construct for caspase 9 was restriction digested with NciI, and caspase 9 cRNA (32P-labeled) was produced using the BD PharMingen in vitro transcription system and SP6 RNA polymerase (Sigma). To produce cRNA for caspase 9b, the riboprobe construct for caspase 9b was restriction digested with BamHI, and caspase 9b cRNA (32P-labeled) was produced using the BD PharMingen in vitro transcription system. The Bcl-x(L/s) riboprobe template was part of the hAPO2 multitemplate set from BD PharMingen and labeled following the standard protocol.

Ribonuclease Protection Assays-- Total RNA (5 µg) from A549 cells was hybridized to 500,000 cpm of 32P-labeled cRNA probe using the BD PharMingen ribonuclease protection assay system. RNase-protected fragments were produced following the manufacturer's protocol. Protected RNA fragments were resolved on 5% PAGE-7 M urea gels (Bio-Rad), dried at 80 °C for 1 h, and autoradiograms produced using Bio-Max film (Kodak).

Protein Extraction-- Total protein was extracted by direct lysis with Laemmli buffer. Cells were lysed with 0.1 ml of 2 × Laemmli buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.04% bromphenol blue, and 250 mM beta -mercaptoethanol) after resuspension in 0.1 ml of ice-cold phosphate-buffered saline (PBS). Samples were boiled for 10 min and either examined directly by SDS-polyacrylamide gel electrophoresis or stored at -20 °C.

Western Immunoblotting-- Total protein lysate (20 µg) was subjected to 6% (PARP), 12% (caspase 9/9b), or 15% (Bcl-x(L/s) SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) and blocked in 5% milk, 1 × PBS-T (M-PBS-T) for 2 h. The membrane was incubated with anti-PARP (Santa Cruz), anti-Bcl-x IgG (Santa Cruz (P19)), or anti-caspase 9 IgG (Stressgen) for 2 h in M-PBS-T followed by 3 washes with PBS-T. The membrane was then incubated with a secondary antibody of horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody for 45 min followed by 3 washes with PBS-T. Immunoblots were developed using Amersham ECL reagents and Bio-Max film.

Quantification of Ceramide Levels: Pulse Labeling with [3H]palmitic Acid-- 2 × 106 A549 cells were incubated with 1 µCi/ml [3H]palmitic acid (16.7 nM) with simultaneous addition of gemcitabine. After 24 h, lipids were extracted from the radiolabled cells using the Bligh-Dyer method as described (52). Ceramide levels were measured following TLC analysis and normalized to total lipid phosphate as described (53, 54).

MTT Assay-- 1.5 × 104 A549 cells were plated into each well of a 96-well plate in a 50-µl volume. After 24 h at standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37 °C), the cells were treated with the appropriate concentration of D-e-C6 ceramide or gemcitabine in a 50-µl volume and returned to the incubator. After the appropriate time, 25 µl of MTT solution (5 mg/ml) was added and cells were again incubated under standard conditions for 5 h. Cells were then lysed and solubilized by the addition of 100 µl of lysis solution (20% SDS (w/v), 50% dimethyl formamide (v/v), and 0.8% acetic acid). The plate was read at A595.

Statistical Analysis-- Tests were done in triplicate on at least two separate occasions. The standard deviations and standard error of mean were determined after quantitating a change in the ratio of one alternative splice variant versus another following treatment as compared with untreated and sham control samples. It was determined that a two-way ANOVA at p < 0.05 to be significant. All statistical tests were done with StatView software (SSI Corp.) and Microsoft EXCEL.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exogenous Ceramide Regulates the Alternative Splicing of Bcl-x and Caspase 9 Pre-mRNA-- We have previously reported that SR proteins, a family of factors that regulate alternative splicing, are dephosphorylated in a time- and dose-dependent manner in response to ceramide (49). Since many apoptosis-regulating factors have alternative splice variants with antagonistic function, ceramide was examined for effects on the pre-mRNA processing of Bcl-x, caspase 9, Bax, and caspase 2. Using an RT-PCR-based assay (Fig. 1, A and B), it was found that overnight treatment of A549 lung adenocarcinoma cells with 20 µM D-e-C6 ceramide (sub-IC50 dose for a 24-h period in A549 cells) (IC50 = 37 µM) resulted in altering the ratio of the splice variants of Bcl-x and caspase 9 (Fig. 2, A and B), but not Bax or caspase 2 (data not shown). In the case of Bcl-x, there was a decrease in the ratio of Bcl-x(L)/Bcl-x(s) from 9.1 to 4.3 (Fig. 2A), and in the case of caspase 9, there was an increase in the ratio of caspase 9/caspase 9b from 6.5 to 22.7 (Fig. 2B).


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  		Fig. 1.   Structure of Bcl-x and caspase 9 genes. A, the schematic depicts the gene structure of Bcl-x, which is composed of three exons and two introns. The Bcl-x(L)- and Bcl-x(s)-specific 5' splice sites are designated. Arrows depict the location of PCR primers used in the RT-PCR assay. The figure also depicts the PCR fragments obtained from the RT-PCR assay and the protected RPA fragments obtained in assaying Bcl-x(L) and Bcl-x(s) mRNA levels. B, the schematic depicts the gene structure of caspase 9 with the exon 3, 4, 5, and 6 cassettes specific for caspase 9 mRNA. Arrows depict the location of PCR primers used in the RT-PCR assay. The figure also depicts the PCR fragments obtained from the RT-PCR assay and the protected RPA fragments obtained in assaying caspase 9 and caspase 9b mRNA levels.


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  		Fig. 2.   The effect of exogenous ceramide on Bcl-x 5' splice site selection and caspase 9 alternative splicing. A, the effect of exogenous ceramide on Bcl-x 5' splice site selection. A549 cells were treated for 24 h with 20 µM D-e-C6 ceramide. Total RNA was extracted and analyzed by RT-PCR and RPA. The graphs depict the ratio of Bcl-x(L) mRNA to Bcl-x(s) mRNA or caspase 9 mRNA to caspase 9b mRNA as determined by densitometry of RT-PCR fragments stained ethidium bromide. Data are expressed as the mean ± S.E. The RT-PCR results are representative of six separate determinations on three separate occasions. The RPA results are representative of three separate determinations on two separate occasions. B, the effect of exogenous ceramide on caspase 9 alternative splicing. Total RNA was extracted and analyzed by RT-PCR and RPA. The graphs depict the ratio of Bcl-x(L) mRNA to Bcl-x(s) mRNA or caspase 9 mRNA to caspase 9b mRNA as determined by densitometry of RT-PCR fragments stained ethidium bromide. Data are expressed as the mean ± S.E. The RT-PCR results are representative of six separate determinations on three separate occasions. The RPA results are representative of three separate determinations on two separate occasions. C, the effect of exogenous ceramide on the mRNA expression of the constitutive genes, L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Total RNA was extracted and analyzed by RPA and relative changes in mRNA levels determined by densitometry of the corresponding bands generated by autoradiography. The RPA results are representative of three separate determinations on two separate occasions.

Since the RT-PCR-based assay is semiquantitative and assesses only changes in mRNA ratios between the splice variants, a ribonuclease protection assay (RPA) was used to quantitate the individual increases or decreases of each splice variant. Using the RPA assay, caspase 9 mRNA was found to be increased ~9-fold following ceramide treatment with 64.5% decrease in caspase 9b mRNA (Fig. 2B). For Bcl-x, Bcl-x(L) mRNA was found to be decreased 42% and Bcl-x(s) mRNA which was undetectable in control cells was now detectable following ceramide treatment (Fig. 2A). The RPA was normalized to L32 and glyceraldehyde-3-phosphate dehydrogenase mRNAs (Fig. 2C).

The effects on mRNA levels translated to the protein level as ceramide treatment decreased the immunoreactive protein levels of Bcl-x(L) by 67% with a concomitant 2.5-fold increase in the immunoreactive protein levels of Bcl-x(s) (Fig. 3A). Similarly at the protein level, ceramide induced an increase in the immunoreactive protein levels of caspase 9 and a concomitant decrease in the immunoreactive protein levels of caspase 9b (Fig. 3B). Thus, since ceramide increases one splice variant followed by a decrease in the other splice variant, these data demonstrate that ceramide affects the alternative splicing of caspase 9 and Bcl-x in a manner that promotes apoptosis.


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  		Fig. 3.   The effect of exogenous ceramide on the immunoreactive protein levels of the gene products of Bcl-x and caspase 9. A, effect of ceramide on the immunoreactive protein levels of Bcl-x. A549 cells were treated with 20 µM D-e-C6 ceramide for 24 h, and total protein lysates were produced. Total protein lysates (20 µg) were subjected to 12 and 15% SDS-PAGE analysis, transferred to polyvinylidene difluoride, and immunoblotted for either Bcl-x(s) or Bcl-x(L)/Bcl-xgamma . Bcl-x(s) was 21 kDa and Bcl-x(L)/Bcl-xgamma was 29 and 32 kDa. B, effect of ceramide on the immunoreactive protein levels of caspase 9. A549 cells were treated with 20 µM D-e-C6 ceramide for 24 h, and total protein lysates were produced. Total protein lysates (20 µg) were subjected to 12% SDS-PAGE analysis, transferred to polyvinylidene difluoride, and immunoblotted for caspase 9/9b. Caspase 9/9b was 45 and 32 kDa. Data are representative of three separate determinations reproduced on two separate occasions.

The effects of ceramide on the pre-mRNA processing of Bcl-x and caspase 9 were time- and dose-dependent. For caspase 9, an increase in the caspase 9/caspase 9b ratio was noticeable by 12 h, and 5 µM D-e-C6 ceramide induced profound effects on the alternative splicing of caspase 9, increasing the ratio of caspase 9/caspase 9b to 14.6 with a maximal increase of 22.7 at 20 µM after 24 h (Fig. 4, A and B). In contrast, ceramide did not affect the alternative splicing of Bcl-x until after 16 h of ceramide treatment with maximal effects at 36 h (Fig. 4C). Similar to caspase 9, at least 5 µM D-e-C6 ceramide was necessary to induce a significant decrease in the ratio of Bcl-x(L)/Bcl-x(s) (Fig. 4D). Therefore, the effect of ceramide treatment of the alternative splicing of caspase 9 and Bcl-x is dose- and time-dependent.


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  		Fig. 4.   The time and dose dependence of exogenous ceramide treatment on the alternative splicing of Bcl-x and caspase 9. A, caspase 9 time course. A549 cells were treated with 20 µM D-e-C6 ceramide or EtOH vehicle control for 3, 6, and 12 h. Total RNA was extracted and analyzed by RT-PCR for the alternative splicing of caspase 9. Data are representative of four separate determinations on two separate occasions. B, caspase 9 dose response. A549 cells were treated with various doses (1, 5, 10, and 20 µM) of D-e-C6 ceramide for 24 h. Total RNA was extracted and analyzed by RT-PCR for the alternative splicing of caspase 9. Data are representative of four separate determinations on two separate occasions. C, Bcl-x time course. A549 cells were treated with 20 µM D-e-C6 ceramide or EtOH vehicle control for 12, 16, 24, and 36 h. Total RNA was extracted and analyzed by RT-PCR for the alternative splicing of caspase 9. Data are representative of four separate determinations on two separate occasions. D, Bcl-x dose response. A549 cells were treated with various doses (1, 5, 10, and 20 µM) of D-e-C6 ceramide for 24 h. Total RNA was extracted and analyzed by RT-PCR for the alternative splicing of Bcl-x. Data are representative of four separate determinations on two separate occasions.

Ceramide-induced Alternative Splicing Is Inhibited by Calyculin A, but Not Okadaic Acid-- Previously, the dephosphorylation of SR proteins in response to the generation of endogenous ceramide was found to be dependent on PP1, a ceramide-activated protein phosphatase (49). To establish whether a ceramide-activated protein phosphatase plays a role in regulating the alternative splicing of caspase 9 and Bcl-x, we pretreated A549 cells for 2 h with 5 nM calyculin A, an inhibitor of both PP1 and PP2A-type protein phosphatases. Calyculin A completely blocked the ceramide effects on caspase 9 and Bcl-x alternative splicing (Fig. 5, A and B). To establish whether PP1 or PP2A was the ceramide-responsive protein phosphatase regulating caspase 9 and Bcl-x alternative splicing, A549 cells were pretreated for 2 h with 10 nM okadaic acid, a selective PP2A inhibitor at this dose in cells (49, 55, 56). Pretreatment with okadaic acid had no effect on either caspase 9 or Bcl-x alternative splicing (Fig. 5, A and B). Calyculin A, but not okadaic acid also inhibited the effect of ceramide on the immunoreactive levels of caspase 9 and Bcl-x. Taken together, these results suggest that PP1 mediates the effects of ceramide on the alternative splicing of both caspase 9 and Bcl-x.


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  		Fig. 5.   Effects of inhibitors of serine/threonine-protein phosphatases and fumonisin B1 on the activation of the Bcl-x(s) 5' splice site and the alternative splicing of caspase 9. A, the effect of inhibitors of serine/threonine-protein phosphatases on the alternative splicing of caspase 9. A549 cells were pretreated 2 h with either 10 nM okadaic acid or 5 nM calyculin A followed by 24 h treatment with 20 µM D-e-C6 ceramide. Total RNA was extracted and analyzed by RT-PCR for ceramide-induced alternative splicing. Data are representative of three separate determinations on two separate occasions. B, the effect of inhibitors of serine/threonine-protein phosphatases on the activation of the Bcl-x(s) 5' splice site. A549 cells were pretreated 2 h with either 10 nM okadaic acid or 5 nM calyculin A followed by 24 h treatment with 20 µM D-e-C6 ceramide. Total RNA was extracted and analyzed by RT-PCR for ceramide-induced alternative splicing. Data are representative of three separate determinations on two separate occasions. C, effect of fumonisin B1 on ceramide-induced activation of the Bcl-x(s) 5' splice site. A549 cells were pretreated 2 h with 100 µM fumonisin B1 followed by 24 h treatment with 20 µM D-e-C6 ceramide. Total RNA was extracted and analyzed by RT-PCR, respectively, for ceramide-induced alternative splicing. Data are representative of four separate determinations on three separate occasions. D, effect of fumonisin B1 on ceramide-induced alternative splicing of caspase 9. A549 cells were pretreated 2 h with 100 µM fumonisin B1 followed by 24 h treatment with 20 µM D-e-C6 ceramide. Total RNA was extracted and analyzed by RT-PCR, respectively, for ceramide-induced alternative splicing. Data are representative of four separate determinations on three separate occasions.

Ceramide-induced Alternative Splicing Is Inhibited by Fumonisin B1, an Inhibitor of CoA-dependent Ceramide Synthase-- Exogenous ceramide treatment has been demonstrated to increase endogenous ceramide via deacylation/reacylation in A549 cells.2 A key enzyme in this pathway is CoA-dependent ceramide synthase, and fumonisin B1 is a highly specific fungal inhibitor of this enzyme (57, 58). Since the dephosphorylation of SR proteins in response to exogenous ceramide treatment was found to be dependent on endogenous ceramide generated via CoA-dependent ceramide synthase (49), we determined whether exogenous cell-permeable ceramides acted directly or whether endogenous ceramide mediated the effects of exogenous ceramide on the alternative splicing of caspase 9 and Bcl-x. Pretreatment of A549 cells were for 2 h with 100 µM fumonisin B1, a dose established to block CoA-dependent ceramide synthase in A549 cells, completely inhibited both caspase 9 and Bcl-x alternative splicing in response to ceramide (Fig. 5, C and D). Thus, endogenous ceramide generated through the action of the CoA-dependent ceramide synthase is implicated in regulating the alternative splicing of Bcl-x and caspase 9 pre-mRNA.

Endogenous Ceramide Generated by the de Novo Sphingolipid Pathway Induces the Alternative Splicing of Caspase 9 and Bcl-x-- Since CoA-dependent ceramide synthase is an enzyme in the de novo biosynthetic pathway of ceramide, we established a model in A549 cells for generation of de novo ceramide in response to extracellular agents. The de novo synthesis of ceramide was assessed directly by pulse labeling A549 cells with [3H]palmitic acid. Treatment of A549 cells with the chemotherapeutic drug, gemcitabine (1 µM), for 24 h induced a 3.1-fold increase in [3H]ceramide (Fig. 6A). Pretreatment with myriocin, a specific inhibitor of serine palmitoyltransferase (the first enzyme in sphingolipid biosynthesis), blocked the increase in [3H]ceramide following 24 h of gemcitabine exposure (Fig. 6A). Furthermore, pretreatment of A549 cells with myriocin (50 nM), reduced the basal levels of [3H]ceramide by 62% (Fig. 6A). Thus, the increase in ceramide levels in response to gemcitabine occurs via the de novo sphingolipid pathway in A549 cells.


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  		Fig. 6.   The effects of gemcitabine treatment on the levels of ceramide and the alternative splicing of Bcl-x and caspase 9. A, the effect of gemcitabine on the levels of [3H]ceramide following pulse labeling with [3H]palmitate. A549 cells were pretreated 2 h with or without 50 nM myriocin followed by a 24-h treatment with 1 µM gemcitabine and 16.7 nM [3H]palmitate. Total lipids were extracted using the Bligh-Dyer method, and the extracted lipids were base hydrolyzed, subjected to thin layer chromatography (TLC), and autoradiographed. The results are presented as arbitrary densitometry units of labeled ceramide. Samples were normalized to nanomoles of total lipid phosphate prior to TLC. Data are representative of three separate determinations reproduced on two separate occasions. B, the effect of gemcitabine on the alternative splicing of caspase 9. A549 cells were treated for 24 h with 1 µM gemcitabine. Total RNA was extracted and analyzed by RT-PCR for gemcitabine-induced alternative splicing. Data are representative of three separate determinations on two separate occasions. C, the effect of gemcitabine on the alternative splicing of Bcl-x. A549 cells were treated for 24 h with 1 µM gemcitabine. Total RNA was extracted and analyzed by RT-PCR for gemcitabine-induced alternative splicing. Data are representative of three separate determinations on two separate occasions.

To establish that ceramide generated via the de novo sphingolipid pathway will induce a change in the alternative splicing of caspase 9 and Bcl-x, we again treated A549 cells with gemcitabine for 24 h. Using the RT-PCR based assay, we demonstrated that treatment with gemcitabine (1 µM) induced an increase in the ratio of caspase 9 mRNA/caspase 9b mRNA from 5.1 to 19.2. Furthermore, the ratio of Bcl-x(L) to Bcl-x(s) was decreased significantly from 8.7 to 3.8 (Fig. 6, B and C). To demonstrate that this mechanism was dependent on the generation of ceramide via the de novo sphingolipid pathway, A549 cells were pretreated with myriocin (50 nM). Pretreatment with myriocin blocked gemcitabine-induced alternative splicing (Fig. 7, A and B). Thus, the alternative splicing of caspase 9 and Bcl-x are regulated by the generation of endogenous ceramide via the de novo sphingolipid pathway.


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  		Fig. 7.   The effects of myriocin on gemcitabine-induced alternative splicing of caspase 9 and Bcl-x on the viability of A549 cells. A, the effect of myriocin on gemcitabine-induced alternative splicing of caspase 9. A549 cells were pretreated for 30 min with 50 nM myriocin followed by treatment with 1 µM gemcitabine for 24 h. Total RNA was extracted and analyzed by RT-PCR for gemcitabine-induced alternative splicing. Data are representative of three separate determinations on two separate occasions. B, the effect of myriocin on gemcitabine-induced alternative splicing of Bcl-x. A549 cells were pretreated for 30 min with 50 nM myriocin followed by treatment with 1 µM gemcitabine for 24 h. Total RNA was extracted and analyzed by RT-PCR for gemcitabine-induced alternative splicing. Data are representative of three separate determinations on two separate occasions.

Ceramide-induced Alternative Splicing Occurs Prior to Apoptotic Cell Death and Sensitizes A549 Cells to Daunorubicin-- To determine whether the increase in pro-apoptotic caspase 9 and Bcl-x(s) and decrease in anti-apoptotic caspase 9b and Bcl-x(L) occurs prior to apoptosis, we determined the time course of apoptosis following treatment with gemcitabine. Treatment of A549 cells for 24 h with gemcitabine (1 µM) did not induce significant cell death as measured by MTT assay and there was no observed cleavage of PARP following these treatments (Fig. 8). Significant cell death was not observed until after 72 h of treatment (58%) by MTT assay, and PARP cleavage was also not observed until after 72 h (Fig. 8). Thus, the change in the alternative splicing of caspase 9 and Bcl-x occurs prior to apoptosis induced by gemcitabine in A549 cells.


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  		Fig. 8.   The effects of gemcitabine on the viability of A549 cells. A, the effect of gemcitabine on the viability of A549 cells after 24 h. A549 cells were examined in the presence or absence of 1 µM gemcitabine for 24 h by MTT assay as described under "Materials and Methods." Data are presented as % control viability. Data are representative of three separate determinations on two separate occasions. B, the effect of gemcitabine on the viability of A549 cells after 72 h. A549 cells were examined in the presence or absence of 1 µM gemcitabine for 72 h by MTT assay as described under "Materials and Methods." Data are presented as % control viability. Data are representative of three separate determinations on two separate occasions. C, the effect of gemcitabine on PARP proteolysis. A549 cells were treated with 1 µM gemcitabine for 24 and 72 h, and total protein lysates were produced. Total protein lysates (20 µg) were subjected to 12 and 15% SDS-PAGE analysis, transferred to polyvinylidene difluoride, and immunoblotted for PARP. Unproteolysed PARP is 115 kDa and cleaved PARP is 85 kDa. Data are representative of four separate determinations reproduced on two separate occasions.

To determine whether ceramide-induced alternative splicing may play a role in sensitization of cells to chemotherapy, we treated A549 cells with various doses of daunorubicin in the presence of either vehicle, 1, 5, 10, or 20 µM D-e-C6 ceramide. Treatment of A549 cells with 5, 10, or 20 µM D-e-C6 ceramide significantly lowered the IC50 of daunorubicin from 3.7 µM to 0.65, 0.425, and 0.425 µM, respectively (Table I). Treatment of A549 cells with a dose of D-e-C6 ceramide (1 µM) that has no effect on the alternative splicing of caspase 9 and Bcl-x, had no effect on the IC50 of daunorubicin (Table I). Thus, ceramide-induced alternative splicing of caspase 9 and Bcl-x correlates with the sensitization of A549 cells to daunorubicin.

                              
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Table I
The effect of ceramide treatment on the effectiveness of daunorubicin to induce apoptosis in A549 cells
A549 cells were treated with 0, 0.001, 0.005, 0.010, 0.050, 0.075, 0.100, 0.250, 0.500, 0.750, 1.000, 2.500, 5.000, 7.500, 10.000, 15.000, and 20.000 µM daunorubicin in the presence of the indicated amounts of ceramide for 48 h, assayed for mitochondrial function using an MTT assay, and the daunorubicin IC50 determined and depicted. Data are presented as mean IC50 value and is representative of four separate determinations reproduced on two separate occasions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recently, we reported that ceramide, a known inducer of apoptosis, regulated the phosphorylation state of SR proteins, a family of RNA transactivating factors that regulate alternative splicing (49). Based on these previous findings and on the observations that several protein factors which regulate apoptosis have splice variants with a dominant/negative function, we examined whether the processing of Bcl-x, caspase 9, Bax, and caspase 2 pre-mRNA was affected by ceramide. The results from this study demonstrate that ceramide induces, via alternative splicing, the levels of pro-apoptotic splice variants Bcl-x(s) and caspase 9, with a concomitant loss in the anti-apoptotic splice variants, Bcl-x(L) and caspase 9b. This newly defined and novel mechanism was also demonstrated to be dependent on both PP1 activation and endogenous ceramide. These observations are important for several reasons. First, this mechanism of ceramide-induced alternative splicing defines a novel mechanism of regulating the gene expression of pro-apoptotic factors in response to extracellular agents. Second, a direct and specific mechanism mediated by a ceramide-activated protein phosphatase and endogenous ceramide generated via the de novo sphingolipid pathway has been defined. Third, a new mechanism involved in the initiation stage of apoptosis and the sensitization of cells to chemotherapy has been implicated.

Previous studies on agonist-induced alternative splicing have mainly focused on mitogenic pathways activated by hormones and growth factors such as insulin and insulin-like growth factor 1 which have been shown to regulate the alternative splicing of the insulin receptor, protein kinase Cbeta , and protein-tyrosine phosphatase-1 (45, 59-66). To our knowledge, the results in this study describe, for the first time, a novel mechanism by which apoptotic stimuli can affect gene expression through alternative splicing. In this case, ceramide, a mediator of apoptosis, reduced the expression of the cell survival factors caspase 9b and Bcl-x(L) and increased the expression of the pro-apoptotic factors Bcl-x(s) and caspase 9. This was demonstrated by a semi-quantitative assay based on RT-PCR which demonstrated a change in the ratio of caspase 9 to caspase 9b and in the ratio of Bcl-x(L) to Bcl-x(s). To verify that both splice variants were being affected over the time periods presented, we used a ribonuclease protection assay that was highly quantitative. Using this assay, Bcl-x(L) was shown to decrease with ceramide treatment (following the published half-life of ~14 h for Bcl-x(L) mRNA) as well as caspase 9b while caspase 9 and Bcl-x(s) were both demonstrated to increase significantly (67, 68). Since both splice variants are affected in response to ceramide, transcriptional effects are an unlikely mechanism of action as both splice variants would be affected in the same direction.

The mechanisms that regulate the gene expression of either caspase 9 or Bcl-x are of direct relevance to the apoptotic mechanism as both caspase 9 and Bcl-x have been demonstrated to be important mediators of apoptosis. More specifically, the differential regulation of apoptosis by isoforms of both Bcl-x and caspase 9 generated via alternative splicing is of importance as overexpression of either caspase 9 or Bcl-x(s) induces apoptosis, whereas overexpression of caspase 9b and Bcl-x(L) has been shown to inhibit apoptosis in response to Fas, tumor necrosis factor alpha , Bax, TRAIL, UV radiation, and Bik (11, 12, 75-81). Moreover, recent reports have demonstrated a role for the alternative splicing of Bcl-x in chemotherapy sensitivity and induction of apoptosis. These studies reported that exposure of A549 cells (24 h) to an oligonucleotide that specifically interacted and blocked the 5' splice site for Bcl-x(L) within exon 2, induced down-regulation of Bcl-x(L) with a concomitant increase in Bcl-x(s) (69, 70). Simultaneously, the IC50 of daunorubicin for A549 cells was decreased by 80% (69, 70). Furthermore, chronic exposure (>24 h) to the oligonucleotide-induced apoptosis (71). Thus, by affecting the alternative splicing of Bcl-x pre-mRNA, cells become more sensitive to chemotherapy and undergo apoptosis. This mechanism would promote apoptosis by increasing cell death components with the simultaneous attenuation of cell survival factors (10-12, 72-74). Thus, the current studies define a novel mechanism of regulating the alternative splicing of regulators of apoptosis in response to ceramide.

Of interest, Bcl-x(L) mRNA still predominated after ceramide treatment. Thus the question is raised as to whether enough Bcl-x(s) is expressed to overcome normal Bcl-x(L) expression in cells. Thompson and co-workers (10) demonstrated that only 1 molecule of Bcl-x(s) per 4 molecules of Bcl-x(L) was necessary to overcome the Bcl-x(L) survival mechanism. Thus, in this study, ceramide lowered the ratio of Bcl-x(L)/Bcl-x(s) mRNA from 9.3 to 4.1. This was reflected at the protein level by a decrease in Bcl-x(L) and an increase in Bcl-x(s) immunoreactive protein. If the mRNA ratio reflects the protein ratio of Bcl-x(L)/Bcl-x(s), this new ratio of Bcl-x(L)/Bcl-x(s) would promote apoptosis. A similar case was demonstrated for activation of caspase 9. Alnemri and co-workers (12) showed that only 1 protein molecule of caspase 9b to 4 protein molecules of caspase 9 completely blocked the activation of caspase 9 in vitro. In the current study, ceramide enhanced the ratio of caspase 9/caspase 9b mRNA from 5:1 (95% inactive caspase 9 in vitro) to almost 20:1 (fully active caspase 9 in cells). Interestingly, the protein levels of caspase 9b were higher than caspase 9 which did not correspond to the mRNA ratio of caspase 9 to caspase 9b in A549 cells as determined by RT-PCR. On the other hand, the protein levels of caspase 9 and 9b were reflected closely by the RPA assays which are more quantitative. This is explained by the high GC-rich content of the caspase 9b mRNA/cDNA which lowers its extension rate compared with caspase 9 mRNA/cDNA during PCR by 80% (data not shown). Since a ratio for caspase 9/caspase 9b mRNA cannot be established using the presented RPA data, an additional role for ceramide regulation of either translational efficiency of the caspase 9b mRNA or different protein stabilities between caspase 9 and caspase 9b cannot be completely ruled out.

Mechanistically, ceramide-induced alternative splicing was dependent on the activation of PP1. This conclusion was based on the use of the potent inhibitors of serine/threonine-protein phosphatases, okadaic acid, and calyculin A. In this study, we demonstrate that the PP1 and PP2A inhibitor, calyculin A, completely blocked ceramide-induced alternative splicing of Bcl-x and caspase 9. On the other hand, okadaic acid, a specific inhibitor of PP2A at the concentrations used, had no effect on ceramide-induced alternative splicing of Bcl-x and caspase 9. This therefore infers that the mechanism is dependent of PP1 activation. This conclusion is supported by several previous reports. First, natural ceramides have been shown to activate PP1 in a stereospecific manner in vitro (37). Second, activation of PP1 in cells has been demonstrated to occur in response to the generation of endogenous ceramide via CoA-dependent ceramide synthase and via the de novo sphingolipid pathway. Third, the dephosphorylation of a family of RNA splice factors, SR proteins, has been demonstrated to require the activation of PP1 (49). Finally, this conclusion is further supported by a report from Lamond and co-workers (48) who demonstrated that dephosphorylation of SR proteins with PP1 induced alternative 5' splice site selection in vitro. This correlates with Bcl-x alternative splicing, which is determined by alternative selection of 5' splice sites within the Bcl-x exon 2. These data, therefore, suggest that the alternative splicing of both caspase 9 and Bcl-x are regulated by ceramide via activation of a ceramide-activated protein phosphatase, in particular, PP1.

The role of endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was also examined since we had already established with PP1 that endogenous ceramide regulated the phosphorylation state of SR proteins. Treatment with short chain ceramides has been shown to increase ceramide levels in A549 cells via a CoA-dependent ceramide synthase in a fumonisin B1-inhibitable manner. Fumonisin B1 also blocked ceramide-induced alternative splicing demonstrating the dependence on endogenous ceramide. To establish more conclusively the role of endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x, a model of de novo ceramide was established in A549 cells. Gemcitabine treatment was demonstrated to increase the endogenous ceramide levels in A549 cells via the activation of de novo ceramide synthesis. This conclusion is based on the observation that myriocin, an inhibitor of the generation of de novo ceramide, totally prevented the increase in endogenous ceramide in response to gemcitabine. Myriocin also blocked the effects of gemcitabine on the alternative splicing of Bcl-x and caspase 9. Thus, de novo ceramide was shown to regulate the alternative splicing of Bcl-x and caspase 9. In another cell model, Molt-4 acute T-cell leukemia cells, etoposide treatment also induces a change in the alternative splicing of Bcl-x pre-mRNA (data not shown) and etoposide has been demonstrated to increase primarily de novo ceramide (54). These data correlate with our previous report of de novo ceramide regulating the dephosphorylation of SR proteins (49). The role of SR proteins in ceramide-induced alternative splicing is currently under investigation.

The studies presented suggest a role for ceramide-induced alternative splicing in sensitizing cells to apoptosis. As previously discussed, inducing an increase in Bcl-x(s) by directly targeting the 5' splice site specific for Bcl-x(L) leads to increased chemotherapeutic sensitivity and eventually to apoptosis in A549 cells (69-71). Thus, the mechanism of ceramide-induced alternative splicing may have direct relevance to drug resistance. Our results are in accord with this hypothesis demonstrating that only doses of D-e-C6 ceramide that affect the alternative splicing of caspase 9 and Bcl-x sensitize A549 cells to daunorubicin. Furthermore, gemcitabine induces a splicing change in Bcl-x and caspase 9 within 24 h, but does not induce significant apoptosis until 72 h post-treatment. Therefore, ceramide-induced alternative splicing may play a role in producing a pro-apoptotic phenotype with enhanced sensitization of cells to apoptotic stimuli. Studies are under way to investigate this possibility as well as the possible role in chemotherapy resistance.

In conclusion, these results demonstrate a novel mechanism in which endogenous ceramide and PP1 regulate the alternative splicing of specific apoptotic factors, caspase 9 and Bcl-x. This mechanism may have direct relevance to the action of chemotherapeutic agents that function to induce intracellular levels of ceramide, such as etoposide, Ara-C, Taxol, gemcitabine, and daunorubicin (Fig. 9) (29, 54, 75-81).


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  		Fig. 9.   Hypothetical schematic of the signal transduction pathway mediating the alternative splicing of Bcl-x and caspase 9.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA87584 and GM43825 (to Y. A. H.), National Research Service Award GM19953-02 (to C. E. C.) from the National Institutes of Health, and a VA Merit Review (to C. E. C.) from the Veterans Administration.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, Rm. 501, Basic Science Building, Medical University of South Carolina, 173 Ashley Ave., P.O. Box 250509, Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322; E-mail: hannun@musc.edu.

Published, JBC Papers in Press, January 18, 2002, DOI 10.1074/jbc.M112010200

2 Ogretmen, B., Pettus, B. J., Rossi, M. J., Wood, R., Usta, J., Szulc, Z., Bielawska, A., Obeid, M., and Hannun, Y. A. (2002) J. Biol. Chem. in press.

    ABBREVIATIONS

The abbreviations used are: RT, reverse transcriptase; RPA, ribonuclease protection assay; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PBS, phosphate-buffered saline; EtOH, ethanol; cRNA, complementary RNA; PARP, poly(ADP-ribose) polymerase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Clarke, P. G., and Clarke, S. (1996) Anat. Embryol. (Berl.) 193, 81-99[Medline] [Order article via Infotrieve]
2. Kerr, J. F. R., and Harmon, B. V. (1991) in Apoptosis: The Molecular Basis of Cell Death (Tomei, L. D. , and Cope, F. O., eds) , pp. 5-29, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
3. Kerr, J. F. R., and Wyllie, A. H. (1992) Intl. Rev. Cytol. 68, 251-306
4. Kerr, J. F., Winterford, C. M., and Harmon, B. V. (1994) Cancer 73, 2013-2026[CrossRef][Medline] [Order article via Infotrieve]
5. Tewari, M., Quan, L. T., K, O. R., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Cell 81, 801-809[CrossRef][Medline] [Order article via Infotrieve]
6. Reed, J. C. (1995) Hematol. Oncol. Clin. N. Am. 9, 451-473[Medline] [Order article via Infotrieve]
7. Reed, J. C. (1997) Adv. Pharmacol. 41, 501-532[Medline] [Order article via Infotrieve]
8. Schendel, S. L., Xie, Z., Montal, M. O., Matsuyama, S., Montal, M., and Reed, J. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5113-5118[Abstract/Free Full Text]
9. Miyashita, T., and Reed, J. C. (1992) Cancer Res. 52, 5407-5411[Abstract/Free Full Text]
10. Minn, A. J., Boise, L. H., and Thompson, C. B. (1996) J. Biol. Chem. 271, 6306-6312[Abstract/Free Full Text]
11. Seol, D. W., and Billiar, T. R. (1999) J. Biol. Chem. 274, 2072-2076[Abstract/Free Full Text]
12. Srinivasula, S. M., Ahmad, M., Guo, Y., Zhan, Y., Lazebnik, Y., Fernandes-Alnemri, T., and Alnemri, E. S. (1999) Cancer Res. 59, 999-1002[Abstract/Free Full Text]
13. Jiang, Z. H., Zhang, W. J., Rao, Y., and Wu, J. Y. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9155-9160[Abstract/Free Full Text]
14. Apte, S. S., Mattei, M. G., and Olsen, B. R. (1995) Genomics 26, 592-594[CrossRef][Medline] [Order article via Infotrieve]
15. Priault, M., Camougrand, N., Chaudhuri, B., and Manon, S. (1999) FEBS Lett. 443, 225-228[CrossRef][Medline] [Order article via Infotrieve]
16. Schmitt, E., Paquet, C., Beauchemin, M., Dever-Bertrand, J., and Bertrand, R. (2000) Biochemistry 270, 868-879
17. Shi, B., Triebe, D., Kajiji, S., Iwata, K. K., Bruskin, A., and Mahajna, J. (1999) Biochem. Biophys. Res. Commun. 254, 779-785[CrossRef][Medline] [Order article via Infotrieve]
18. Thomas, A. L., Price, C., Martin, S. G., Carmichael, J., and Murray, J. C. (1999) Cell Death Differ. 6, 97-108[CrossRef][Medline] [Order article via Infotrieve]
19. Zhou, M., Demo, S. D., McClure, T. N., Crea, R., and Bitler, C. M. (1998) J. Biol. Chem. 273, 11930-11936[Abstract/Free Full Text]
20. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769-1771[Abstract/Free Full Text]
21. Zhang, J., Alter, N., Reed, J. C., Borner, C., Obeid, L. M., and Hannun, Y. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5325-5328[Abstract/Free Full Text]
22. Obeid, L. M., and Venable, M. E. (1997) J. Am. Geriatr. Soc. 45, 361-366[Medline] [Order article via Infotrieve]
23. Jayadev, S., Liu, B., Bielawska, A. E., Lee, J. Y., Nazaire, F., Pushkareva, M., Obeid, L. M., and Hannun, Y. A. (1995) J. Biol. Chem. 270, 2047-2052[Abstract/Free Full Text]
24. Gamard, C. J., Dbaibo, G. S., Liu, B., Obeid, L. M., and Hannun, Y. A. (1997) J. Biol. Chem. 272, 16474-16481[Abstract/Free Full Text]
25. Hannun, Y. A., and Obeid, L. M. (1997) Biochem. Soc. Trans. 25, 1171-1175[Medline] [Order article via Infotrieve]
26. Venable, M. E., Lee, J. Y., Smyth, M. J., Bielawska, A., and Obeid, L. M. (1995) J. Biol. Chem. 270, 30701-30708[Abstract/Free Full Text]
27. Liu, B., Andrieu-Abadie, N., Levade, T., Zhang, P., Obeid, L. M., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 11313-11320[Abstract/Free Full Text]
28. Smyth, M. J., Perry, D. K., Zhang, J., Poirier, G. G., Hannun, Y. A., and Obeid, L. M. (1996) Biochem. J. 316, 25-28[Medline] [Order article via Infotrieve]
29. Ogretmen, B., Schady, D., Usta, J., Wood, R., Kraveka, J. M., Luberto, C., Birbes, H., Hannun, Y. A., and Obeid, L. M. (2001) J. Biol. Chem. 276, 24901-24910[Abstract/Free Full Text]
30. Kim, M. Y., Linardic, C., Obeid, L., and Hannun, Y. (1991) J. Biol. Chem. 266, 484-489[Abstract/Free Full Text]
31. Zhang, P., Liu, B., Jenkins, G. M., Hannun, Y. A., and Obeid, L. M. (1997) J. Biol. Chem. 272, 9609-9612[Abstract/Free Full Text]
32. Dbaibo, G. S., Perry, D. K., Gamard, C. J., Platt, R., Poirier, G. G., Obeid, L. M., and Hannun, Y. A. (1997) J. Exp. Med. 185, 481-490[Abstract/Free Full Text]
33. Bielawska, A., Greenberg, M. S., Perry, D., Jayadev, S., Shayman, J. A., McKay, C., and Hannun, Y. A. (1996) J. Biol. Chem. 271, 12646-12654[Abstract/Free Full Text]
34. Law, B., and Rossie, S. (1995) J. Biol. Chem. 270, 12808-12813[Abstract/Free Full Text]
35. Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523-15530[Abstract/Free Full Text]
36. Wolff, R. A., Dobrowsky, R. T., Bielawska, A., Obeid, L. M., and Hannun, Y. A. (1994) J. Biol. Chem. 269, 19605-19609[Abstract/Free Full Text]
37. Chalfant, C. E., Kishikawa, K., Mumby, M. C., Kamibayashi, C., Bielawska, A., and Hannun, Y. A. (1999) J. Biol. Chem. 274, 20313-20317[Abstract/Free Full Text]
38. Misteli, T., and Spector, D. L. (1996) Mol. Biol. Cell 7, 1559-1572[Abstract]
39. Zahler, A. M., Lane, W. S., Stolk, J. A., and Roth, M. B. (1992) Genes Dev. 6, 837-847[Abstract/Free Full Text]
40. Zahler, A. M., Neugebauer, K. M., Lane, W. S., and Roth, M. B. (1993) Science 260, 219-222[Abstract/Free Full Text]
41. Zahler, A. M. (1999) Methods Mol. Biol. 118, 419-432[Medline] [Order article via Infotrieve]
42. Manley, J. L., and Tacke, R. (1996) Genes Dev. 10, 1569-1579[Free Full Text]
43. Du, C., McGuffin, M. E., Dauwalder, B., Rabinow, L., and Mattox, W. (1998) Mol. Cell 2, 741-750[CrossRef][Medline] [Order article via Infotrieve]
44. Caceres, J. F., Misteli, T., Screaton, G. R., Spector, D. L., and Krainer, A. R. (1997) J. Cell Biol. 138, 225-238[Abstract/Free Full Text]
45. Du, K., Peng, Y., Greenbaum, L. E., Haber, B. A., and Taub, R. (1997) Mol. Cell. Biol. 17, 4096-4104[Abstract]
46. Lin, C. H., and Patton, J. G. (1995) Rna 1, 234-245[Abstract]
47. Lopato, S., Mayeda, A., Krainer, A. R., and Barta, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3074-3079[Abstract/Free Full Text]
48. Mermoud, J. E., Cohen, P. T., and Lamond, A. I. (1994) EMBO J. 13, 5679-5688[Medline] [Order article via Infotrieve]
49. Chalfant, C. E., Ogretmen, B., Galadari, S., Kroesen, B. J., Pettus, B. J., and Hannun, Y. A. (2001) J. Biol. Chem. 276, 44848-44855[Abstract/Free Full Text]
50. Boudrez, A., Beullens, M., Groenen, P., Van Eynde, A., Vulsteke, V., Jagiello, I., Murray, M., Krainer, A. R., Stalmans, W., and Bollen, M. (2000) J. Biol. Chem. 275, 25411-25417[Abstract/Free Full Text]
51. Hirano, K., Erdodi, F., Patton, J. G., and Hartshorne, D. J. (1996) FEBS Lett. 389, 191-194[CrossRef][Medline] [Order article via Infotrieve]
52. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917[Medline] [Order article via Infotrieve]
53. Perry, D. K., Bielawska, A., and Hannun, Y. A. (2000) Methods Enzymol. 312, 22-31[Medline] [Order article via Infotrieve]
54. Perry, D. K., Carton, J., Shah, A. K., Meredith, F., Uhlinger, D. J., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 9078-9084[Abstract/Free Full Text]
55. Lee, J. Y., Hannun, Y. A., and Obeid, L. M. (1996) J. Biol. Chem. 271, 13169-13174[Abstract/Free Full Text]
56. Kishikawa, K., Chalfant, C. E., Perry, D. K., Bielawska, A., and Hannun, Y. A. (1999) J. Biol. Chem. 274, 21335-21341[Abstract/Free Full Text]
57. Merrill, A. H., Jr., van Echten, G., Wang, E., and Sandhoff, K. (1993) J. Biol. Chem. 268, 27299-27306[Abstract/Free Full Text]
58. Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T., and Merrill, A. H., Jr. (1991) J. Biol. Chem. 266, 14486-14490[Abstract/Free Full Text]
59. Wilson, G. M., Sun, Y., Sellers, J., Lu, H., Penkar, N., Dillard, G., and B